Thp 1

Ten different human CRC cell lines, CaR-1, RKO, Colo205, Colo320DM, DLD1, HCT116, Lovo, SW480, SW620 and HT29 and the human monocyte line THP-1 were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University. They were maintained in RPMI 1640 supplemented with 10% FBS at 37°C in a 5% humidified CO₂ atmosphere.
To assess if the production of cellular factors such as exosomes from miR-203-transfected cells affected the differentiation of THP-1, co-culture experiments were carried out in a transwell culture plate (Corning-Costar, Acton, MA, USA) allowing free exchange of culture medium and solutes. miR-203-transfected cells or the control and THP-1 were seeded in the lower and upper wells, respectively.

Leukemia lines MOLM13, OCIAML2, THP1, U937, MV411, HL60, SEM, TF1, and HEL were maintained in RPMI-1640 medium (CORNING, 10–040-CV) containing 10% fetal bovine serum (FBS) (HyClone, 03.N16001DC). HEK293T cells were cultured in DMEM (HyClone, SH300022.01) supplemented with 10% FBS and 4.5-mM L-glutamine. The OCIAML2 and SEM cell lines were originally purchased from DSMZ. MOLM13 was originally purchased from ACCEGEN. The THP1, U937, MV411, and HL60 cell lines were originally purchased from the American Type Culture Collection (ATCC). The TF1 and HEL cell lines were obtained from Cell Resource Center, Peking Union Medical College (PCRC). The HEK293T cells (no. SCSP-502) were obtained from the Cell Bank of the Chinese Academy (www.cellbank.org.cn). Before using experiments, cell line identity was verified by STR analysis, and cells were confirmed as mycoplasma negative.

Cells of the human monocytic cell line THP-1 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells and THP-1 cells transformed with an mRFP-GFP-LC3B reporter (provided by Hongbo Shen, Institute Pasteur of Shanghai, China) were maintained in RPMI 1640 (Corning) culture medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Life Technologies) at 37°C and 5% CO₂. The THP-1 cells were seeded at 5 × 10⁵ cells/ml in a 12-well plate in complete RPMI 1640 and differentiated using phorbol 12-myristate 13-acetate (PMA; Sigma, P8139) at 40 ng/ml for 24 h. Following removal of the PMA-containing medium, the cells were incubated in the fresh complete RPMI 1640 medium for 12 h at 37°C for further use.
Bazedoxifene (acetate) (BZA) was purchased from MedChemExpress (MCE; China), and a 10 mM stock solution was dissolved and diluted in dimethyl sulfoxide (DMSO; Sigma, USA). Bafilomycin A1 (Baf), N-acetyl-l-cysteine (NAC), and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (Sigma, USA). The final concentration of DMSO in treatment did not exceed 0.1% (vol/vol).

Both the human KIRC cell line Caki-1 and the human macrophage line THP-1 were purchased from American Type Culture Collection(ATCC company). THP-1 cells and Caki-1 cells were all cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone™) supplemented with 10% fetal bovine serum (FBS) (Gibco™) at 37°C with 5% CO₂ in a humidified incubator. To assess the interaction between macrophages and RCC cells, Caki-1 cells and THP-1 cells were co-cultured in a Transwell system (0.4 μm pore diamete, Corning Incorporation) in a 6-well plate for 24 h, allowing free diffusion of molecules between the two compartments but not cell translocation. THP-1 cells were seeded in the upper chamber of Transwell system and co-cultured with Caki-1 cells in the bottom chamber.

BEAS-2B and THP-1 cells were obtained from ATCC (Manassas, VA, USA). BEAS-2B cells were suspended in BEGM medium at a density of 1 × 10⁵/mL, and then added in 96-well plates with 100 μL/well. Aliquots of 3 × 10⁴ THP-1 cells were seeded in 0.1 mL RPMI 1640 complemented with 1 μg/mL phorbol 12-myristate acetate (PMA) in 96-well plates (Corning, NY, USA). After overnight incubation at 37 °C, the culture media were replaced with MO suspensions in BEGM or RPMI 1640 media at concentrations ranging from 0 to 200 μg/mL, followed by 24 h incubation. Then the ATP or MTS assay solutions were added to evaluate cell viabilities by measuring the luminescence or absorbance on a SpectraMax M5 microplate spectrophotometer.

Cell culture and cytokine profiling were performed as described previously (26 (link), 32 (link)). The human monocyte cell line THP-1 (ATCC, Manassas, VA) was cultured in RPMI 1640 (Corning) supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO₂ incubator. THP-1 cells were adjusted to 5 × 10⁵ viable cells/mL and placed into fresh medium with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO) to induce differentiation into a macrophage-like state. After 48 h of incubation and cell washing, sterile 0.4-mm transwell inserts were placed in the wells with THP-1 cells and the inserts were filled with 125 mL of medium and P. gingivalis W83 parent strain or derivatives. After 2, 6, and 24 h, the cell culture supernatant fluids were collected and the levels of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, IL-10, and RANTES were determined by using Milliplex multiplex assays (EMD, Millipore, Billerica, MA). Data were acquired on a Luminex 200 system running xPONENT 3.1 software (Luminex, Austin, TX) and analyzed using a 5-parameter logistic spline curve-fitting method using Milliplex Analyst version 5.1 software (Vigene Tech, Carlisle, MA).